In healthcare, there is a trend towards the development of so-called Point Of Care (POC) devices, which are small devices, often with disposable components such as cartridges, that can be used in diagnosis and treatment of patients as an alternative to large and expensive analysis equipment.
A widely used diagnostic test is a Full blood count (FBC) test, which is a diagnostic test that is used to measure cellular composition of blood. It may give information about the status of an immune system of a patient, about the ability of the blood to disseminate oxygen and/or about the ability of the blood to effectively clot. As such, it is a fundamental test that is often used as an initial “general purpose” diagnostic tool or as a more targeted monitoring solution. Examples of care cycles that include a full blood count as a monitoring tool include oncology, arthritis and Crohn's disease. As many as 300 million FBC tests are performed each year in the developed world.
Currently, large scale commercial laboratory instruments known as haematology analyzers are used to automatically perform all measurements that comprise the FBC. The high cost and complexity of these devices, coupled to the need for venous blood, means that they are mostly large scale, centralized facilities. There is a clear clinical need for performing FBC in a near patient setting, particularly for applications that require a full blood count to monitor the progression and/or treatment of a disease.
Previously, microfluidic point of care devices have been developed which are capable of measuring individual components of the FBC. In that area, Hb measuring devices, WBC counters capable of performing a white blood cell differential and platelet count devices, devices which optically count and determine size of red blood cells are available. For cell counting, current haematology analyzers typically employ electrical coulter counting and/or optical scattering methods to count and differentiate white cells and to count and determine size of the red blood cells and platelets.
At the moment only few examples of micro fluidic coulter counter technologies exist. One example combines a coulter counter with a Hb measurement. Another example of counting cells is by flow-through impedance spectroscopy. This is a flow cytometry analysis which is especially suited for a micro fluidic format. This technique is capable of differentiating between lymphocytes, monocytes and neutrophils in lysed blood, and of counting and sizing red blood cells and platelets.
The current “gold-standard” for Hb measurement is the photometric cyanmethaemoglobin (HbCN) method disclosed in Standardization of hemoglobinometry II, The hemiglobincyanide method, Clin Chim Acta, 1961, 6, p. 38-44. This method involves chemical lysis of the red blood cells and subsequent labelling of all the Hb that these cells release with a cyanide ion. The labels produce a defined absorption profile with a maximum at 540 nm. By measuring the optical absorption at 540 nm, the concentration of Hb can be determined. Furthermore, the high stability of HbCN means that it is easy to supply a calibration standard.
The most common red blood cell lysis/cyanide conversion reagent is known as Drabkin's reagent. Drabkin's reagent contains Potassium Cyanide, which is extremely toxic. This reagent only works for very large dilutions in whole blood (1:251), since red blood cell lysis relies on the low ionic strength of the reagent to induce osmotic shock. This large dilution causes an inherent imprecision in the method. Furthermore, to measure the optical absorption at 540 nm, very long optical path lengths of ˜1 cm are required. Finally, in some pathological samples, turbidity can lead to erroneously high absorption readings, which in turn will give rise to an incorrect Hb concentration.
To avoid the problems associated with toxicity and turbidity, many other optical means of measuring Hb have been developed. A known point of care device uses sodium azide to convert the Hb to an azide-coordinated Hb derivative (azidemethemoglobin, HbN3). This method itself lends to short path length (0.1 mm) absorption spectroscopy, since dry reagents remove the need for dilution of the whole blood. Two absorbance readings are taken to determine the HbN3 concentration, i.e. one at the absorption maximum (565 nm) and one at 800 nm to correct for turbidity.
For the point of care WBC/Hb counter, a RBC lysis solution has been developed that preserves the WBCs while at the same time labeling the Hb molecule with imidazole. In a similar way as described above, the optical absorption of the imidazole labeled Hb species is measured at two wavelengths, i.e. one at the absorption peak and one to correct for turbidity and scattering effects for the white blood cells. The same solution may also be passed through a coulter counter to perform the cell count.
Another known lysis/Hb conversion reagent is based on sodium lauryl sulphate/sodium dodecyl sulphate (SLS/SDS). The SDS lyses all the blood cells and labels the Hb to get an SDS-coordinated derivative. Since SDS is a surfactant molecule, turbidity correction is not necessary and so a single absorption reading at 535 nm is taken to determine the Hb concentration. This method is designed for high dilutions of Hb, so the inherent imprecision present in the HbCN measurement is still present in the HbSDS one.
All the above described devices and techniques are capable of performing specific measurements from a finger-prick of blood. However, none of the above described devices and techniques are capable of measuring all parameters that are required for an FBC in a single POC measurement. Recently, a microfluidic device capable of performing a FBC in a single POC measurement has been disclosed in WO 2010/086786. This microfluidic device comprises a two sample preparation stages, one for diluting a portion of a blood sample with a lysis agent for a white blood cell count and a quench solution and providing the diluted portion to an impedance measurement means and a second dilution stage for diluting a further portion of the blood sample with a diluent for haemoglobin measurement and providing the diluted further portion to a measurement means for determining properties of red blood cells, such as RBC count, HB count and platelet count. The diluent is fed to the blood sample several times to obtain a high dilution ratio. Consequently, only a fraction of the RBC count sample is used for the actual RBC count, with well over 90% of the various dilution stages being fed to waste.
It is of paramount importance that the flow rates through such a microfluidic device are well-defined in order to achieve accurate measurement results of the FBC. Such flow rates may be controlled using separate pumps for each fluid stream, but this is rather costly. Alternatively, the flow rates may be well-defined (i.e. tuned) at the design stage of the microfluidic device by tuning the dimensions (i.e. the fluidic resistance) of the fluid channels forming the microfluidic network. As the feature sizes of the microfluidic network are typically larger than the feature sizes of e.g. the impedance measurement chip, it is easier and therefore more cost-effective to manufacture the microfluidic network and the measurement chip in separate processes.
This, however, complicates the tuning of the microfluidic device. For instance, as only part of the diluted blood sample is fed to the measurement chip, the remainder of the diluted blood sample is typically fed to waste, as previously explained. Due to the larger feature sizes of the microfluidic network compared to the impedance measurement chip, the waste channel in the microfluidic network typically has to comprise a fluidic resistance matching element to ensure that ratio of the fluidic resistance of the waste channel and the measurement channel through the impedance measurement chip is well-defined and comparable.
It has however been found that this matching element does not always achieve satisfactory tuning. This is because the tolerances of the manufacturing process of the microfluidic network are independent of the tolerances of the manufacturing process of the impedance measurement chip, such that the required dimensions of the matching element cannot be accurately predicted.